Aggregation of Polyglutamine-expanded Ataxin 7 Protein Specifically Sequesters Ubiquitin-specific Protease 22 and Deteriorates Its Deubiquitinating Function in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex

Abstract

Human ataxin 7 (Atx7) is a component of the deubiquitination module (DUBm) in the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex for transcriptional regulation, and expansion of its polyglutamine (polyQ) tract leads to spinocerebellar ataxia type 7. However, how polyQ expansion of Atx7 affects DUBm function remains elusive. We investigated the effects of polyQ-expanded Atx7 on ubiquitin-specific protease (USP22), an interacting partner of Atx7 functioning in deubiquitination of histone H2B. The results showed that the inclusions or aggregates formed by polyQ-expanded Atx7 specifically sequester USP22 through their interactions mediated by the N-terminal zinc finger domain of Atx7. The mutation of the zinc finger domain in Atx7 that disrupts its interaction with USP22 dramatically abolishes sequestration of USP22. Moreover, polyQ expansion of Atx7 decreases the deubiquitinating activity of USP22 and, consequently, increases the level of monoubiquitinated H2B. Therefore, we propose that polyQ-expanded Atx7 forms insoluble aggregates that sequester USP22 into a catalytically inactive state, and then the impaired DUBm loses the function to deubiquitinate monoubiquitinated histone H2B or H2A. This may result in dysfunction of the SAGA complex and transcriptional dysregulation in spinocerebellar ataxia type 7 disease.

Keywords: DUB module; SAGA complex; USP22; aggregation; ataxin 7; deubiquitylation (deubiquitination); polyglutamine; protein-protein interaction; sequestration; zinc finger.

FIGURE 1.

PolyQ-expanded Atx7 sequesters USP22 into inclusions in cells as visualized by confocal microscopy. A, sequestration of overexpressed USP22. Atx7_10Q-GFP or Atx7_EPQ-GFP (100Q) was cotransfected with FLAG-USP22 into HEK 293T cells, and the cells were visualized by confocal fluorescence microscopy. Atx3_71Q-GFP and FLAG-USP5 were set as controls. USP22 and USP5 were stained with anti-FLAG antibody (red), and nuclei were stained with Hoechst (blue). Scale bar =10 μm. B, sequestration of endogenous USP22. HEK 293T cells transfected with Atx7_10Q-GFP or Atx7_EPQ-GFP (100Q) were visualized by confocal fluorescence microscopy. USP22 was stained with anti-USP22 antibody (red), and nuclei were stained with Hoechst (blue). Scale bar = 10 μm.

FIGURE 2.

Characterization of the interaction between Atx7 and USP22. A, the domain architecture of Atx7 and USP22. Qn, polyQ tract; PRR, proline-rich region; ZnF, zinc finger domain; SCA7, SCA7 domain; USPD, ubiquitin-specific protease domain. B, co-IP experiment for the interaction between the N-terminal fragment of Atx7 and endogenous USP22. HEK 293T cells were transfected with FLAG-Atx7_10Q-N and FLAG-Atx7_EPQ-N (115Q), respectively, and the cell lysates were subjected to coimmunoprecipitation with anti-FLAG antibody. USP22 was detected with an anti-USP22 antibody. Vec, vector. C, co-IP experiment for the interacting fragment in USP22. HA-Atx7_10Q-N was cotransfected with FLAG-tagged USP22 or its fragments into HEK 293T cells, and the cell lysates were subjected to coimmunoprecipitation with anti-FLAG antibody. USP22-N, residues 1–170; USP22-C, residues 172–525. D, GST pulldown experiment showing that Atx7-ZnF interacts directly with the N-terminal ZnF domain of USP22. Top panel, SDS-PAGE graph with Coomassie Blue (CBB) staining. Bottom panel, Western blotting (WB) to detect GB1-His₆-tagged Atx7-ZnF with anti-His antibody (10% input). Atx7-ZnF, residues 75–172 of Atx7; USP22-N, residues 1–170 of USP22 containing a ZnF domain.

FIGURE 3.

Characterizing the role of the ZnF domain in Atx7 in interaction with and sequestration of USP22 by mutagenesis. A, sequence for the ZnF domain of Atx7 showing the mutation sites. The three ZnF mutants are as follows: C2*, C150S/C153S; H2*, H165N/H170N; and C2H2*, C150S/C153S + H165N/H170N. B, co-IP experiment for the interaction of Atx7_10Q-N or its mutants with endogenous USP22. HEK 293T cells were transfected with FLAG-Atx7_10Q-N or its mutants, and the cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. V, vector. C, sequestration of endogenous USP22 by Atx7_EPQ or its ZnF mutants as visualized by confocal microscopy. Atx7_EPQ-GFP or its ZnF mutants were transfected into HEK 293T cells. The polyQ lengths of Atx7_EPQ-GFP used were as follows: WT, 100Q; C2*, 109Q; H2*, 90Q; C2H2*, 99Q. USP22 was stained with anti-USP22 antibody (red), and nuclei were stained with Hoechst (blue). Scale bar = 10 μm.

FIGURE 4.

PolyQ-expanded Atx7 sequesters endogenous USP22 into aggregates. A, sequestration of endogenous USP22 into insoluble aggregates by Atx7_EPQ-N. FLAG-tagged Atx7_10Q-N or Atx7_EPQ-N (115Q) was transfected into HEK 293T cells, and the cell lysates were subjected to fractionation and Western blot analysis with an anti-FLAG or anti-USP22 antibody. Sup, supernatant; Pel, pellet; Vec, vector. B and C, dose-dependent experiments for the sequestration of USP22 into aggregates by Atx7_10Q-N (B) or Atx7_EPQ-N (C). FLAG-tagged Atx7_10Q-N or Atx7_EPQ-N (115Q) was transfected into HEK 293T cells in a dose-dependent manner. D, time course experiments for the sequestration of USP22 into aggregates by Atx7_EPQ-N. FLAG-tagged Atx7_EPQ-N (115Q) was transfected into HEK 293T cells, and the cell lysates were subjected to fractionation in a time course manner. E, quantification of endogenous USP22 and Atx7_EPQ-N in pellets (data from D). Data are mean ± S.E. (n = 3). F, comparison of the sequestration of USP22 by Atx7_EPQ-N and its ZnF mutants. FLAG-tagged Atx7_EPQ-N and its mutants were transfected into HEK 293T cells, respectively. The polyQ lengths of Atx7_EPQ-N used were as follows: WT, 115Q; C2*, 115Q; H2*, 115Q; C2H2*, 106Q.

FIGURE 5.

Detection of the catalytic activities of USP22 immunoprecipitated by Atx7-N variants. A, Ub-VS modification of catalytically active USP22 immunoprecipitated by Atx7_10Q-N and Atx7_EPQ-N (100Q). HEK 293T cells were transfected with FLAG-tagged Atx7_10Q-N or Atx7_100Q-N, and then the cell lysates were subjected to immunoprecipitation. The immunoprecipitated species were incubated with or without Ub-VS (5 μm), and the reaction mixtures were detected by Western blotting. B, quantification of the ratio of USP22^Ub-VS against total USP22 (data from A). Data are mean ± S.E. (n = 3). **, p < 0.01.

FIGURE 6.

Effects of various Atx7-N forms on cellular H2Bub levels. A, assay of the H2Bub level affected by Atx7_10Q-N or Atx7_EPQ-N. FLAG-tagged Atx7_10Q-N or Atx7_EPQ-N (115Q) was transfected into HeLa cells, and the cell lysates were subjected to Western blotting with an anti-H2Bub or anti-H2B antibody. Vec, vector. B, quantification of the H2Bub levels affected by Atx7-N forms. Data are mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; N.S., no significance. C, as in A, by Atx7_EPQ-N and its ZnF mutants. The polyQ lengths of Atx7_EPQ-N used were as follows: WT, 115Q; C2*, 115Q; H2*, 115Q; C2H2*, 106Q. D, quantification of the H2Bub levels affected by Atx7_100Q-N and its ZnF mutants. Data are mean ± S.E. (n = 3). **, p < 0.01. E, assay of the H2Bub level affected by full-length Atx7. Various HA-Atx7-GFP forms were transfected into HeLa cells, and the cell lysates were subjected to Western blotting with an anti-H2Bub or anti-H2B antibody. C2H2*, 99Q. F, quantification of the H2Bub levels affected by full-length Atx7. Data are mean ± S.E. (n = 3). *, p < 0.05.

FIGURE 7.

Schematic of the aggregation of polyQ-expanded Atx7 impairing the deubiquitination of H2Bub. PolyQ-expanded Atx7 forms aggregates that sequester USP22 into a catalytically inactive state in the DUB module. The impaired DUB module loses the function to deubiquitinate monoubiquitinated histone H2B or H2A. The polyQ tract of Atx7 is highlighted in red. Atx7, USP22, Atx7L3, and ENY2 are the components of the DUBm in the SAGA complex.