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. 2013 Nov;33(22):4461-72.
doi: 10.1128/MCB.00480-13. Epub 2013 Sep 16.

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation Through Noncanonical Ubiquitination

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Free PMC article

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation Through Noncanonical Ubiquitination

Sarah M Ronnebaum et al. Mol Cell Biol. .
Free PMC article

Abstract

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging.

Figures

Fig 1
Fig 1
SirT6 protein is stabilized in the presence of CHIP. (A) SirT6 expression was analyzed via Western blot analysis in multiple tissues from 12-month-old CHIP+/+ and CHIP−/− mice. Skel., skeletal. (B) Endogenous SirT6 and CHIP protein expression was measured in shControl and shCHIP 293 cells (left) and in CHIP+/+ and CHIP−/− MEFs (right) via Western blot analysis. SirT6 protein quantification (middle) and mRNA quantification (bottom) are shown (n = 4). (C) Normal 293 cells were transiently transfected with control siRNA, two independent siRNAs targeting CHIP, or SirT6 siRNA. CHIP and SirT6 protein were measured via Western blot analysis, and SirT6 protein quantification (middle) and mRNA quantification (bottom) are shown (n = 3). (D) shControl and shCHIP 293 cells stably expressing FLAG-SirT6 were exposed to dimethyl sulfoxide (DMSO), actinomycin, chloroquine, cycloheximide, MG132, or ammonium chloride (NH4Cl) for 16 h. SirT6 protein levels were visualized in Western blots, quantified, and normalized to the DMSO control for each cell type (n = 3). (Right) Representative Western blot from cells treated with MG132. The proteasome-regulated protein p27 is shown to indicate MG132 effectiveness. (E) shCHIP cells were exposed to DMSO or MG132 for 16 h, and Endogenous SirT6 protein levels were measured via Western blotting. p27 indicates MG132 treatment. (Top) Representative Western blot. (Bottom) Quantification (n = 4). Results represent means and SEM. *, P < 0.05. Graphed results represent SirT6 protein relative to β-tubulin and normalized to control in arbitrary units.
Fig 2
Fig 2
CHIP and SirT6 stably interact through the CHIP U-box domain. (A) HEK 293 cells were transfected with Myc-tagged CHIP (Myc-WT CHIP) and FLAG-tagged sirtuin isoforms 1 to 7 (FLAG-SirT). Input (above) and FLAG immunoprecipitation (IP) (below) were performed on cell lysates and visualized via Western blotting with the indicated antibodies. (B) Endogenous CHIP was immunoprecipitated from HEK 293 cell lysates, and endogenous SirT6 was detected and visualized via Western blotting. (C) HEK 293 cells transfected with Myc-WT CHIP and FLAG-SirT6 were incubated at 37°C or 42°C for 30 min. Nuclear fractions were immediately prepared, and input and Myc immunoprecipitations were visualized via Western blotting with the indicated antibodies. (D) The indicated Myc-tagged CHIP constructs (diagrammed) were transfected into HEK 293 cells with FLAG-SirT6. Input and Myc immunoprecipitations were performed on cell lysates and visualized via Western blotting. (E and F) shCHIP 293 cells (E) and CHIP−/− MEFs (F) were transfected with Myc-WT CHIP, Myc-ΔU-box CHIP, or Myc-H260Q CHIP. Lysates were collected, and endogenous SirT6 expression was quantified via Western blot analysis (right; n = 4 of each). NS, nonspecific band. The results represent means and SEM. *, P < 0.05. Graphed results represent SirT6 protein relative to β-tubulin and normalized to control in arbitrary units.
Fig 3
Fig 3
CHIP ubiquitinates SirT6 with noncanonical chains at K170. (A) shControl and shCHIP 293 cells stably expressing FLAG-SirT6 were transfected with HA-tagged WT ubiquitin (Ub) or HA-tagged K48R ubiquitin and cultured in MG132 for 16 h. Protein levels of input and FLAG-immunoprecipitated samples were visualized via Western blot analysis. The K48R/WT ubiquitination ratio is shown below (n = 3). (B to E) in vitro ubiquitination reactions were performed using recombinant CHIP, SirT6, and ubiquitin. Reaction mixtures containing WT ubiquitin (B), WT ubiquitin or ubiquitin mutants with the indicated lysines mutated to arginine (C), WT ubiquitin or ubiquitin mutants in which the lysine indicated is the only lysine available for ubiquitination (D), or WT ubiquitin or K48-only ubiquitin with the addition of purified 26S proteasome (E) were used to perform ubiquitination reactions and visualized via Western analysis; representative images of at least 3 experiments are shown. (F) Monoubiquitinated SirT6 was analyzed using LC–MS-MS, and a single ubiquitination modification was discovered at K170. (G) in vitro ubiquitination reactions were performed using CHIP, WT ubiquitin, and WT SirT6 or K170R SirT6. For all ubiquitination reactions, representative images are shown (n = 3). The results represent means and SEM. *, P < 0.05. Graphed results represent SirT6 protein relative to β-tubulin and normalized to control in arbitrary units.
Fig 4
Fig 4
Mutant SirT6 has improved stability in CHIP-depleted cells. (A) shControl and shCHIP cells were cotransfected with green fluorescent protein (GFP) and WT SirT6 or K170R SirT6. Forty-eight hours later, the levels of transfected proteins were visualized via Western blotting, and SirT6 levels were calculated relative to GFP as an internal transfection control (below; n = 3). (B) Pulse-chase assays using 35S-labeled methionine/cysteine were used to measure protein half-lives of WT SirT6 and K170R SirT6 in shControl and shCHIP cells expressing FLAG-tagged SirT6 constructs. Following FLAG immunoprecipitation and autoradiography, protein levels were calculated at the indicated time points. Representative autoradiography images are shown below the corresponding graphs (n = 3). (C) WT SirT6 and K170R SirT6 were transiently expressed to achieve similar protein expression in shControl and shCHIP 293 cells and then split and treated for 16 h with DMSO or MG132. SirT6 protein levels were measured via Western blotting. The accumulation of WT SirT6 or K170R SirT6 caused by MG132 for each cell condition was quantified and expressed relative to DMSO treatment (below; n = 3). The results represent means and SEM; *, P < 0.05. Graphed results represent SirT6 protein relative to β-tubulin and normalized to control in arbitrary units.
Fig 5
Fig 5
CHIP depletion decreases SirT6 and increases H3K9Ac promoter occupancy. (A) H3K9Ac was measured and expressed relative to total H3 via Western blot analysis in shControl and shCHIP 293 cells and in CHIP+/+ and CHIP−/− MEFs. Quantification is shown on the right of each Western blot (n = 3). (B and C) Analysis of H3K9Ac ChIP-seq data from shControl and shCHIP cells was performed by Ingenuity pathway analysis (B) and TRANSFAC (C), using peaks associated with the 5′ regions of annotated genes. (D and E) ChIP-qPCR for endogenous SirT6 (D) and H3K9Ac (E) was performed in shControl cells, shCHIP cells, or shCHIP cells overexpressing WT CHIP (shCHIP+CHIP). Protein enrichment within the promoter regions of the indicated genes was calculated relative to input and normalized to shControl levels (n = 3). (F and G) Real-time PCR was used to measure mRNA in 293 cells used in ChIP studies (F) and livers from CHIP+/+ and CHIP−/− mice (n = 3) (G). (H and I) shControl and shCHIP cells were transfected with vector, WT SirT6, or K170R SirT6 constructs; 48 h later, RNA was isolated. BIRC3 mRNA (H) and LDHB mRNA (I) were measured by qPCR and calculated relative to 18S (n = 3). The results represent means and SEM; *, P < 0.05.
Fig 6
Fig 6
Decreased DNA repair in the absence of CHIP is rescued with SirT6 overexpression. (A) CHIP+/+ (white bars) and CHIP−/− (black bars) MEFs were cultured in DMSO, 3-aminobenzamide (3-AB), camptothecin (CTT), or paraquat (PRQ), and viability was assessed using an MTS assay (n = 3). (B) Following drug treatment, MEFs were fixed and stained for γH2AX and DAPI, and foci of >90 nuclei/condition were quantified. The results were clustered by the number of foci per cell and expressed as percentages of the total distribution. (C) Representative images from γH2AX (green) staining. DAPI (blue) is included as a nuclear stain. (D and E) shControl and shCHIP cells stably expressing empty vector or FLAG-SirT6 were treated with MMS (D) or H2O2 (E) for 6 days. The cells were stained with trypan blue and counted, and the percentage of viable cells was calculated (n = 3). (F and G) shControl and shCHIP cells were transiently transfected with vector, WT SirT6, or K170R SirT6. Forty-eight hours later, the cells were exposed to 200 μM MMS (F) or 400 μM H2O2 (G) for 1 h and then allowed to recover for 30 min. Cells were harvested, and the levels of total RPA and RPA phosphorylated at serines 4 and 8 were measured via Western blotting. Quantification of phosphorylated RPA normalized to total RPA is shown below the representative blots (n = 4 for each). The results represent means and SEM; *, P < 0.05.
Fig 7
Fig 7
Model of SirT6 protein stability in the presence and absence of CHIP. Our data demonstrate that CHIP stably interacts with SirT6 in a manner dependent on CHIP's U-box domain. SirT6 is noncanonically ubiquitinated at K170 by CHIP, which prevents canonical ubiquitination at K170 and stabilizes SirT6. This allows SirT6 to exert KDAC and DNA repair activities. In the absence of CHIP, the availability of K170 allows canonical ubiquitination by another E3 ligase, leading to proteasomal degradation.

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