USP22 regulates cell proliferation by deubiquitinating the transcriptional regulator FBP1

Abstract

Ubiquitin-specific protease 22 (USP22) edits the histone code by deubiquitinating H2A and H2B as part of the mammalian SAGA (Spt-Ada-Gcn5) complex, and is required for transcriptional regulation and normal cell-cycle progression. Here, we show that USP22 affects the expression of p21 by altering far upstream element (FUSE)-binding protein 1 (FBP1) ubiquitination, as ablation of USP22 leads to increased FBP1 ubiquitination and decreased FBP1 protein occupancy at the p21 gene. Surprisingly, increased polyubiquitination of FBP1 does not alter its protein stability, but instead modulates the stable recruitment of FBP1 to target loci. Our results indicate a mechanism by which USP22 regulates cell proliferation and tumorigenesis.

Figure 1

USP22 targets FBP1 for deubiquitination. (A) Scheme for identification of substrates with abundant ubiquitination upon USP22 depletion. (B) Depletion of USP22 leads to increased FBP1 ubiquitination in 293T cells. shControl or shUSP22 293T cells were transfected with HA-Ub, ubiquitinated proteins were precipitated and blotted for FBP1. (C) Overexpression of WT but not catalytically dead (C185S) USP22 decreases FBP1 ubiqutination levels in vivo. The 293T cells were transfected with the indicated vectors, ubiquitinated proteins were purified with an anti-HA-resin, resolved by SDS–PAGE and blotted with the indicated antibodies. The asterisk indicates background bands. USP22 coprecipitates with FBP1. FLAG-FBP1 (D) or endogenous FBP1 (E) was purified, and purified fractions were analysed by immunoblot. Et-Br, ethidium bromide; FBP1, far upstream element (FUSE)-binding protein 1; HA, haemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; Ni-NTA, nickel-nitriloacetic acid; shRNA, short-hairpin RNA; Ub, ubiquitin; USP22, ubiquitin-specific protease 22; WT, wild type.

Figure 2

USP22 activity does not affect FBP1 steady-state level. (A) FBP1 steady-state levels were monitored by immunoblot in USP22-depleted or control cells before and after MG132 treatment. (B) FBP1 and TRF1 steady-state levels in USP22-depleted cells after transfection with the indicated vectors. Immunoblots were performed with the indicated antibodies. FBP1, far upstream element (FUSE)-binding protein 1; USP22, ubiquitin-specific protease 22; WT, wild type.

Figure 3

USP22 modulates Lys 63-linked FBP1 ubiquitination. (A) FLAG-FBP1 was purified from the indicated cells and blotted with the Lys 63-linkage-specific or FLAG (FBP1) antibody. (B) Schematic representation of the HA-ubiquitin expression vectors used in C. (C) Enhanced Lys 63-linked FBP1 polyubiquitination on USP22 depletion. Immunoblot of the HA-precipitated fractions after transfections with the indicated vectors. FBP1, far upstream element (FUSE)-binding protein 1; HA, haemagglutinin; IP, immunoprecipitation; shRNA, short-hairpin RNA; Ub, ubiquitin; USP22, ubiquitin-specific protease 22; WB, western blot; WT, wild type.

Figure 4

USP22 depletion alters the expression of FBP1 target genes. The expression levels of p21, MMP1 and c-Myc in normal human fibroblasts (A) and HeLa cells (B) after the indicated depletions. (C) Schematic representation of the p21 FUSE region. Arrows indicate the region amplified in D. (D) ChIP analyses using the indicated antibodies in control, USP22- or FBP1-depleted HeLa cells. In A, B and D, error bars indicate s.e.m., n=3. Asterisks indicate the statistically significant differences (^*P<0.05, ^**P<0.01) relative to control samples, as determined by Student's two-tailed t-test. (E) Immunoblot analysis of the indicated proteins in nuclear extracts purified from control, USP22- or FBP1-depleted HeLa cells. AU, arbitrary units; ChIP, chromatin immunoprecipitation; FBP1, far upstream element (FUSE)-binding protein 1; FIR, FBP interacting repressor; MMP1, matrix metalloproteinase 1; mRNA, messenger RNA; NHF, normal human fibroblast; USP22, ubiquitin-specific protease 22.

Figure 5

Impaired cell proliferation on USP22 and FBP1 depletion. Impaired proliferation in MCF7 (A) and H1299 (B) cells after depletion of USP22 or FBP1. USP22 or FBP1 depletion leads to p21 upregulation in MCF7 (C) and H1299 (D) cells. Error bars in A–D indicate s.e.m., n=3. Asterisks in C and D indicate the statistically significant differences (P<0.05) between control samples and denoted depletions, on the basis of Student's two-tailed t-test. (E) Ablation of p21 rescues the growth retardation of USP22- or FBP1-depleted MCF7 cells. The data points from two independent experiments were plotted. (F) Efficiency of the USP22, FBP1 and p21 silencing in the cells used in E. FBP1, far upstream element (FUSE)-binding protein 1; mRNA, messenger RNA; shRNA, short-hairpin RNA; USP22, ubiquitin-specific protease 22.