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. 2013 Aug 27;52(34):5746-59.
doi: 10.1021/bi400684q. Epub 2013 Aug 12.

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

Ryan A Henry  1 , Yin-Ming Kuo, Andrew J Andrews
Affiliations

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

Ryan A Henry et al. Biochemistry. .

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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Figures

Figure 1
Figure 1
Determination of steady-state kinetic parameters of CBP- and p300-mediated acetylation of histone H3 when titrating H3. Experiments were performed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37 °C. Assays contained from 1 to 150 nM p300 or 0.5 to 10 nM CBP, with varying concentrations of H3 (0.25–15 μM) and constant (200 μM) acetyl-CoA. Experiments were quenched with 4 vol of TCA and boiled at 95 °C for 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 H3K18. (B) Nonlinear fit of p300 acetylation of histone H3K14. (C) Comparison of the specificity constants (kcat/KmnH) of CBP (black) and p300 (gray) on H3K9, H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300) between CBP and p300 at each site of H3. All quantified sites can be found in Supplemental Figure 1. The apparent kinetic parameters are summarized in Table 2.
Figure 2
Figure 2
Determination of steady-state kinetic parameters of CBP- and p300-mediated acetylation of histone H3 when titrating acetyl-CoA. Experiments were performed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300, 17.5 μM H3, and varying concentrations of acetyl-CoA (1–200 μM). Assays for CBP contained 7 nM CBP, 7.5 μM H3, and varying concentrations of acetyl-CoA (1–200 μM). Experiments were quenched with 4 vol of TCA and boiled at 95 °C for 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 H3K14. (B) Nonlinear fit of p300 acetylation of histone H3K14. (C) Comparison of the specificity constants (kcat/KmnH) of CBP (black) and p300 (gray) on H3K9, H3K14, H3K18, and H3K23. (D) The log of the ratio of specificity (CBP/p300) between CBP and p300 at each site of H3. All quantified sites can be found in Supplemental Figure 2. The apparent kinetic parameters are summarized in Table 3.
Figure 3
Figure 3
Determination of steady-state kinetic parameters of CBP- and p300-mediated acetylation of histone H3/H4 when titrating H3/H4. Experiments were performed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37 °C. Assays contained from 1 to 50 nM p300 or 1 to 22.5 nM CBP, with varying concentrations of H3/H4 (0.2–10 μM) and constant (200 μM) acetyl-CoA. Experiments were quenched with 4 vol of TCA and boiled at 95 °C for 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 H3K18. (B) Nonlinear fit of p300 acetylation of histone H3K18. (C) Comparison of the specificity constants (kcat/KmnH) of CBP (black) and p300 (gray) on H3K9, H3K14, H3K18, and H3K23 and H4K5, H4K8, H4K12, and H4K16. (D) The difference in change in free energy (ΔΔG) between CBP and p300 at each site of H3 and H4. The Y-axis is inverted to more clearly show favorable (−ΔG) changes. All quantified sites can be found in Supplemental Figures 3 and 4. The apparent kinetic parameters are summarized in Table 4.
Figure 4
Figure 4
Determination of steady-state kinetic parameters of CBP- and p300-mediated acetylation of histone H3/H4 when titrating acetyl-CoA. Experiments were performed at 37 °C in 100 mM ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37 °C. Assays for p300 contained 50 nM p300, 7.5 μM H3/H4, and varying concentrations of acetyl-CoA (1–200 μM). Assays for CBP contained 20 nM CBP, 10 μM H3/H4, and varying concentrations of acetyl-CoA (1–200 μM). Experiments were quenched with 4 volumes of TCA and boiled at 95 °C for 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) Nonlinear fit of p300 acetylation of histone H4K16. (C) Comparison of the specificity constants (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 and p300 at each site of H3 and H4. All quantified sites can be found in Supplemental Figures 5 and 6. The apparent kinetic parameters are summarized in Table 5.
Figure 5
Figure 5
Comparison of specificities of CBP and p300 on H3 and H3/H4. (A) The log of the difference of (H3/H4)/H3 for CBP when substrate is limiting. (B) The log of the difference of (H3/H4)/H3 for p300 when substrate is limiting. (C) The log of the difference of (H3/H4)/H3 for CBP when acetyl-CoA is limiting. (D) The log of the difference of (H3/H4)/H3 for p300 when acetyl-CoA is limiting. (E) Summary of specificities (kcat/K1/2nH(app)) of CBP on H3 when substrate (black) or acetyl-CoA (light gray) is limited, or on H3/H4 when substrate (dark gray) or acetyl-CoA (dark gray border) is limited. (F) Summary of specificities (kcat/K1/2nH(app)) of p300 on H3 when substrate (black) or acetyl-CoA (light gray) is limited, or on H3/H4 when substrate (dark gray) or acetyl-CoA (dark gray border) is limited.
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