The stability of histone acetyltransferase general control non-derepressible (Gcn) 5 is regulated by Cullin4-RING E3 ubiquitin ligase

Abstract

Histone acetyltransferases play important roles in the regulation of chromatin structure and gene transcription. As one of the most important histone acetyltransferases, general control non-derepressible (Gcn) 5 has been linked to diverse cellular processes and tumorigenesis as well. We have recently identified a functional link between Gcn5 and acidic nucleoplasmic DNA-binding protein 1 (And-1) that is elevated in multiple cancer cells and is essential for Gcn5 protein stability. However, the mechanism by which And-1 regulates Gcn5 protein stability remains unknown. Here we show that the ablation of Cullin4-RING E3 ubiquitin ligase (CRL4) leads to the stabilization of Gcn5 in cells with depleted And-1, and Cdc10-dependent transcript 2 (Cdt2) serves as a substrate receptor protein of CRL4. Overexpression of Cdt2 reduces the Gcn5 protein levels, and CRL(Cdt2) is sufficient to ubiquitinate Gcn5 both in vivo and in vitro. And-1 stabilizes Gcn5 by impairing the interaction between Gcn5 and CRL(Cdt2) and thereby preventing Gcn5 ubiquitination and degradation. The degradation of Gcn5 is not dependent on proliferating cell nuclear antigen, an important player involved in CRL(Cdt2)-mediated protein degradation. Thus, CRL(Cdt2) and And-1 play an essential role in the regulation of Gcn5 protein stability. This study provides us with the mechanistic basis to develop alternative approaches to inhibit Gcn5 activity for cancer therapy.

FIGURE 1.

Gcn5 is ubiquitinated in vivo and degraded by proteasome. A, depletion of And-1 results in the reduction of Gcn5 proteins and acetylation of histone H3K9 and H3K56 in both HeLa and U2OS cells. Cells treated with siRNA control (siGl2) or two independent siRNAs (siAnd-1-1 or siAnd-1-2) were harvested 48 h after siRNA transfection and subjected to immunoblot using anti-Gcn5, anti-H3K56Ac, anti-H3K9Ac, anti-And-1, or anti-actin antibody. B, treatment of And-1-depleted cells with the proteasome inhibitor MG132 stabilizes Gcn5 proteins. Upper, HeLa cells transfected with the indicated siRNAs were treated with (+) or without (-) MG132 (10 μm) and lysed. Lysates were subjected to immunoblot using anti-Gcn5, anti-And-1, or anti-tubulin antibody. Lower, quantification of Gcn5 protein levels. Means ± S.D. (error bars) were obtained from three independent experiments. The signals of Gcn5 and tubulin proteins were quantified by Scion imaging software, and the background signal was subtracted. The relative levels of Gcn5 proteins were normalized by control tubulin protein signals. The Gcn5 protein levels in siGl2-treated cells alone were taken as 100%. C, Gcn5 is ubiquitinated in vivo. U2OS cells were transfected with HA-ubiquitin and FLAG-Gcn5 or vector, and the ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot using anti-FLAG antibody. IP, immunoprecipitation; Ub, ubiquitin.

FIGURE 2.

Gcn5 forms complexes with CRL4^Cdt2 E3 ligase and And-1. A, Cdt2, DDB1, Cul4A, and And-1 interact with full-length (Full) Gcn5 and Gcn5 mutant (492–837). 293T cells transfected with the indicated FLAG-Gcn5 plasmids were harvested 36 h after transfection for immunoprecipitation using anti-FLAG M2-agarose beads. The FLAG immunoprecipitates (IP) were then subjected to immunoblot (IB) using anti-Cdt2, anti-Cul4A, anti-DDB1, anti-And-1, anti-actin, or anti-FLAG antibody. B, And-1 does not interact with Cdt2. 293T cells transfected with the indicated plasmids were treated as in A. Note that Gcn5 was detected in FLAG-And-1 precipitates.

FIGURE 3.

Down-regulation of CRL4^Cdt2 E3 ligase stabilizes Gcn5 proteins in And-1-depleted cells. A, down-regulation of Cdt2 stabilizes Gcn5 proteins in cells with depleted And-1 by siRNA. Upper, U2OS cells transfected with the indicated siRNAs were harvested and subjected to immunoblot using anti-Gcn5, anti-And-1, anti-Cdt2, or anti-actin antibody. Lower, quantification of Gcn5 protein levels. Means ± S.D. (error bars) were obtained from four independent experiments. The relative levels of Gcn5 proteins were measured as in Fig. 1B. *, nonspecific bands recognized by anti-Cdt2 antibody. B, down-regulation of DDB1 stabilizes Gcn5 in cells with depleted And-1 by siRNA. Upper, U2OS cells transfected with the indicated siRNAs were harvested and subjected to immunoblot using anti-Gcn5, anti-And-1, anti-DDB1, or anti-actin antibody. Lower, quantification of Gcn5 protein levels. Means ± S.D. (error bars) were obtained from four independent experiments. The relative levels of Gcn5 proteins were measured as in Fig. 1B. C, overexpression of Cdt2 results in the reduction of Gcn5 protein levels. U2OS cells transfected with vector or FLAG-Cdt2 plasmids were harvested 36 h after transfection for immunoblot using anti-Gcn5, anti-FLAG, or anti-tubulin antibody. Lower, cells treated as above were harvested for FACS analysis. D, cells treated as in C were harvested for immunofluorescence using anti-FLAG and Gcn5 antibody. FLAG-vector is a plasmid expressing FLAG-And-1 (HMG domain). Note that cells with expression of FLAG-Cdt2 displayed reduced Gcn5 fluorescence signals as compared with cells without expression of FLAG-Cdt2.

FIGURE 4.

PCNA is dispensable for the degradation of Gcn5 by CRL4^Cdt2 E3 ligase. A, Gcn5 does not interact with PCNA. Left, 293T cells transfected with the indicated plasmids were harvested for immunoprecipitation (IP) using anti-FLAG M2-agarose beads. FLAG precipitates were subjected for immunoblot using anti-FLAG, anti-And-1, or anti-PCNA antibody. Note that And-1 was detected in FLAG-Gcn5 precipitates. Right, 293T cells were harvested for immunoprecipitation using anti-Gcn5 antibody or IgG. The Gcn5 precipitates were subjected for immunoblot using anti-And-1, anti-Gcn5, or anti-PCNA antibody. B, down-regulation of PCNA does not stabilize Gcn5 proteins in cells with depleted And-1 by siRNA. Left, U2OS cells transfected with the indicated siRNAs were harvested and subjected to immunoblot using anti-Gcn5, anti-PCNA, anti-And-1, or anti-actin. Right, quantification of Gcn5 protein levels. Means ± S.D. (error bars) were obtained from four independent experiments. The relative levels of Gcn5 proteins were measured as in Fig. 1B.

FIGURE 5.

And-1 prevents the ubiquitination of Gcn5 by CRL4^Cdt2 E3 ligase. A, overexpression of Cdt2 enhances the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested 48 h after transfection. Cells were treated with MG132 for 6 h before harvest. The ubiquitinated species were precipitated by HA antibodies and subjected to immunoblot for ubiquitinated FLAG-Gcn5 using anti-FLAG antibody. Ub, ubiquitin. B, overexpression of And-1 suppresses the ubiquitination of Gcn5. U2OS cells transfected with the indicated plasmids were harvested for 48 h and treated as in A. Note that And-1 overexpression increased the FLAG-Gcn5 protein levels and reduced the ubiquitination of Gcn5. IP, immunoprecipitation; LC, loading control. C, And-1 prevents Gcn5 ubiquitination by CRL4^Cdt2 in vitro. Incubation of immunopurified CRL4^Cdt2 with Gcn5 in an in vitro ubiquitin ligase assay increased the formation of Gcn5 ubiquitinated species. The addition of immunopurified And-1 suppressed the Gcn5 ubiquitination by CRL4^Cdt2. IF, immunofluorescence. D, And-1 overexpression suppresses the ubiquitination of the C terminus of Gcn5. U2OS cells transfected with the indicated plasmids were harvested. FLAG-And-1(1–491) or FLAG-And-1(492–837) was precipitated using anti-FLAG M2-agarose beads. FLAG precipitates and inputs were subjected to immunoblot using anti-ubiquitin, anti-FLAG, or anti-And-1 antibody.

FIGURE 6.

And-1 competes with CRL4^Cdt2 for the association with Gcn5. A, schematic of Gcn5 truncation mutants used for protein-protein interactions as in B. The P300/CBP-associated factor (PCAF) homology domain (PCAF-HD), histone acetyltransferase (HAT) domain, and bromo domain (BD) are indicated. B, And-1 and CRL4^Cdt2 interact with Gcn5 at the same site within the C terminus. 293T cells transfected with the indicated plasmids were harvested and immunoprecipitated using anti-FLAG M2-agarose beads. FLAG-Gcn5 immunoprecipitates (IP) were subjected to immunoblot using anti-FLAG, anti-Cdt2, anti-DDB1, or anti-And-1 antibody. C, And-1 depletion increased the interaction between Gcn5 and CRL4^Cdt2. U2OS cells transfected with the indicated siRNAs and FLAG-Gcn5 plasmid were treated with MG132 to stabilize Gcn5 before harvest. Cells were harvested 55 h after siRNA transfection for immunoprecipitation using anti-FLAG M2-agarose beads. FLAG-Gcn5 precipitates were subjected to immunoblot using anti-And-1, anti-Cdt2, anti-Cul4A, anti-actin, or anti-FLAG antibody. D, And-1 blocks the interaction between Cdt2 and Gcn5 in vitro. Immunopurified Gcn5 proteins on agarose beads were mixed with immunopurified FLAG-Cdt2 in the presence BSA or immunopurified FLAG-And-1 for 3 h at 4 °C. The beads were then washed three times using lysis buffer for immunoprecipitation. Gcn5 precipitates were subjected to immunoblot using anti-FLAG or anti-Gcn5 antibody.

FIGURE 7.

A proposed model for the role of And-1 and CRL4^Cdt2 in the regulation of Gcn5 protein turnover. Both And-1 and CRL4^Cdt2 associate with Gcn5 at the same site within its C terminus. In the presence of And-1, the And-1 module associates with Gcn5 and stabilizes Gcn5 protein levels by preventing the interaction between Gcn5 and CRL4^Cdt2 and thereby the ubiquitination of Gcn5. In the absence of And-1, CRL4^Cdt2 interacts with the C terminus of Gcn5 and ubiquitinates Gcn5, resulting in the degradation of Gcn5. Note that the C-terminal domain of Cul4 associates with a small RING protein regulator of cullin (ROC), which recruits and activates E2 enzyme to transfer ubiquitin (u) to Gcn5.