Autoacetylation of the histone acetyltransferase Rtt109

Abstract

Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 ∼200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation.

FIGURE 1.

Autoacetylation of Rtt109. A, Rtt109 is acetylated at Lys-290, LC MS/MS analysis of Rtt109 recombinantly expressed and purified from E. coli. A representative chromatogram depicts acetylation at Lys-290, with singly charged y-series ions labeled (inset: theoretical b and y fragment ions). Acetylated and deuterated peptides were quantified from an average of three independent purifications. B, deacetylation of Rtt109 by Hst2. Acetyl-lysine immunoblots (top) and SDS-PAGE Coomassie-stained gels (bottom) of Rtt109 and Rtt109 deacetylated by Hst2. Asterisk denotes Rtt109 doublet due to partial C-terminal truncation during the purification. Slightly truncated Rtt109 and full-length Rtt109 are equally acetylated and equally catalytically active (data not shown). C, Rtt109 catalyzes autoacetylation. Deacetylated RV (2 μm) and ^K290QRV (2 μm) were incubated with [¹⁴C]-acetyl-CoA (50 μm) and 20 μl of reactions were resolved on SDS-PAGE gels. Top, [¹⁴C]-acetylated Rtt109 autoradiograph image. Bottom, SDS-PAGE Coomassie-stained gel of the autoacetylation reaction. Asterisk denotes Hst2.

FIGURE 2.

Intramolecular mechanism of autoacetylation and the stimulatory role in histone acetylation. A, intramolecular mechanism of autoacetylation. Log-log plot of the initial rate versus the deacetylated RV concentration. Solid line denotes linear regression curve fit of the data. At least three trials were performed at each deacetylated RV concentration with error bars representing one standard deviation unit. Also shown is the theoretical curve fit (dashed lines) for a second order (slope = 2) reaction for comparison. B, initial rate analysis of autoacetylation in the presence or absence of Rtt109 (already acetylated), deacetylated Rtt109 and acetyl-CoA (50 μm). Experiments were performed three times with error bars representing standard error. C, autoacetylation stimulates HAT activity. The initial rate of histone acetylation from 0–60 s for acetylated RV, deacetylated RV and re-acetylated (reactivated) RV. Assays contained 50 nm RV, 10 μm H3, and 50 μm acetyl-CoA. Experiments were performed three times with error bars representing standard error. N/D*, no detectable rate of acetylation above background (no enzyme).

FIGURE 3.

Thermal denaturation of various Rtt109 constructs and acetyl-CoA autoacetylation saturation curve of deacetylated RV. A, thermal denaturation assays of RV (black), ^K290QRV (dark gray), and ^K290R RV (light gray). A graph with representative experiments is shown. T_m = 50 ± 1 for RV, T_m = 49 ± 1 for ^K290QRV, and T_m = 49 ± 2 for ^K290RRV. B, thermal denaturation assays of Rtt109 (black) and deacetylated Rtt109 (light gray). T_m = 33 ± 1 for Rtt109 and T_m = 33 ± 1 for deacetylated Rtt109. A graph with representative experiments is shown. Error is standard error from at least three independent trials. SDS-PAGE gels of proteins used in thermal melts are in the supplemental data. C, acetyl-CoA saturation curve of the autoacetylation reaction with deacetylated RV (2.5 μm). The apparent K_m = 9 ± 2 μm. Experiments were performed five times with error bars representing one standard deviation unit.

FIGURE 4.

Acetyl-CoA binding curves with RV, ^K290QRV, and ^K290RRV. Intrinsic protein fluorescence measurements with RV constructs. Fluorescence values at 340 nm were utilized to determine K_d. K_d values are 2.7 ± 0.3 for RV, 34 ± 5 μm for ^K290QRV and 95 ± 13 μm for ^K290RRV. A, representative emission spectra of the fluorescence reduction of RV due to acetyl-CoA binding. 0 μm (filled circle), 0.44 μm (x-shape), 0.89 μm (filled diamond), 1.78 μm (open square), 3.56 μm (closed square), 7.11 μm (cross-shape), 14.22 μm (open diamond), 28.44 μm (closed triangle), and 56.90 μm (open circle) of acetyl-CoA was titrated into RV. Acetyl-CoA binding curves with RV (B) ^K290QRV (C), and ^K290RRV(D). At least four independent trials with each RV construct were performed with error bars representing one standard deviation unit.