Thyroid hormone receptor isoform-specific modification by small ubiquitin-like modifier (SUMO) modulates thyroid hormone-dependent gene regulation - PubMed (original) (raw)
Thyroid hormone receptor isoform-specific modification by small ubiquitin-like modifier (SUMO) modulates thyroid hormone-dependent gene regulation
Yan-Yun Liu et al. J Biol Chem. 2012.
Abstract
Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.
Figures
FIGURE 1.
In vitro and in vivo sumoylation of TR. A, [35S]TRα and -TRβ were incubated with E1, UBC9, and SUMO1 in sumoylation buffer at 37 °C for 1 h and then separated by 10% SDS-PAGE. B, mouse liver (L), heart (H), and white adipose (A) tissues were utilized for IP with anti-TRα (rabbit IgG) and anti-TRβ (mouse IgG) antibodies. In WB, anti-full-length SUMO1 was used to detect sumoylation. The membranes shown in B were then stripped, checked by chemiluminescent exposure to confirm the absence of residual activity, and then incubated with anti-TRβ (rabbit IgG) and anti-TRα (rabbit IgG) antibodies (ab; C) to confirm that the identified TR-SUMO bands contained TR. IgG was used as nonspecific control. Arrows indicate the location of TR-SUMO complexes.
FIGURE 2.
Sumoylation of TR isoforms. A, ligand effects and ligase preference for sumoylation HepG2 cells were transfected with human SUMO (SUMO1, 2, or 3)-HA, and human TRα or human TRβ expression vectors and grown in serum-free medium for 16 h prior to treatment with or without T3 (50 n
m
) for 4 h. Cell lysate was used for IP with anti-TRα or anti-TRβ antibodies (ab). Immunoprecipitated protein complexes were subject to 7.5% SDS-PAGE. For detection of TR-SUMO conjugation in WB, anti-HA was used to detect SUMO(1, 2, or 3)-HA. B, E3 ligase requirement. HepG2 cells were transfected with PIAS family members. Control cells were not transfected with E3 ligase. Cells, including control cells, were treated with T3 (50 n
m
) for 4 h for the detection of TRβ-SUMO conjugation, but without T3 for detection of TRα-SUMO. Anti-TRα or anti-TRβ antibodies were used in IP and anti-SUMO1 (full-length) in WB. After IP with anti-TRs, the cell lysates were then used for IP with anti-GAPDH and WB GAPDH, which was used as a protein input control. C, verification of TR in SUMO-conjugated bands in B. The membrane in B was stripped, checked with chemiluminescent exposure to confirm the absence of residual activity, and then incubated with anti-TR antibodies as shown. Arrows show SUMO-TR complexes.
FIGURE 3.
Identification of the sumoylation sites in TRα and TRβ. A, predicted sumoylation sites based on amino acid sequence in TRα and TRβ, with an asterisk showing confirmed sites based on mutation analysis. HepG2 cells were transfected with vectors expressing SUMO1-HA (B; for TRβ) or SUMO3-HA (C; for TRα) and wild type or mutant TRs as shown. Cells were treated with T3 (50 n
m
) for 4 h for TRβ sumoylation, but without T3 for TRα sumoylation. The cell lysate was immunoprecipitated with an anti-HA antibody (ab) to detect SUMO1- or SUMO3-HA, and Western blot was performed with TRβ (B) or TRα (C) antibody. *, confirmed sumoylation sites in TRs. A/B, activation domain; DBD, DNA binding domain; LDB, ligand binding domain.
FIGURE 4.
Sumoylation is important for T3-dependent induction and repression of gene expression. GH3 cells were transfected with siRNA SUMO (1, 2, or 3) or siRNA control using Nucleofactor V, program T-28 (Lonza, Inc.) and plated onto 24-well plates. After 48 h, cells were tested for knockdown efficiency and then cotransfected with expression vectors (TRα or TRβ) and reporter construct rGH-TRE-Luc (A) or rTHSβ-TRE (B) using Effectene transfection reagent (Qiagen). T3 (50 n
m
) was added, and a reporter assay performed after 6 h. C, RT-PCR analysis of SUMO mRNA after siRNA SUMO knockdown and TR mRNA expression in transfected cells. *, T3 induction compared with basal (p < 0.05); **, basal compared with expression without transfected TR and T3 (p < 0.05). C, confirmation of specificity of siRNA is shown with mRNA detection by q-PCR for each SUMO condition and an absence of effect of siRNA on other SUMOs, TRs, or GAPDH.
FIGURE 5.
Effects of sumoylation site TRα and β mutations on T3-mediated gene transcription. Wild-type TRs and TR mutants cotransfected with reporter constructs rGH TRE-luc (A) and TSHβ TRE-luc (B) in HepG2 cells. Cells were grown in serum-replaced medium for 16 h and then treated with T3 (50 n
m
; black bars) or without (white bars) for 4 h prior to luciferase assay. *, with linking bar shows significant effect, p < 0.05 of T3 treatment compared with control; *, compares mutant TR with or without ligand to control, p < 0.05.
FIGURE 6.
The influence of SUMO1 and SUMO3 expression on endogenous rGH mRNA expression and recruitment of TRβ and cofactors to the TRE. A, GH3 cells were transfected with siRNA SUMO1 or SUMO3. Three days after transfection, the medium was changed to serum-replaced medium, and cells were allowed to grow for 16 h. Cells were treated with or without T3 (50 n
m
) for 4 h prior to isolating RNA. The endogenous rGH mRNA expression in GH3 cells was detected by q-PCR. C, control. B, ChIP assays were performed using GH3 cells transfected with TRβ and siRNA SUMO1 or siRNA SUMO3 and antibodies (ab) anti-TRβ, anti-NCoR, and anti-CBP. C, WB analysis of SUMO1 and -3 knockdown. D, the rGH TRE-luc reporter activity was analyzed in TRβ- and TRβ K443Q-transfected GH3 cells with or without addition of 50 n
m
T3. E, ChIP assay of TR binding to rGH TRE and interaction with NCoR and CBP. GH3 cells were transfected with TRβ and TRβ K443Q and treated with or without T3 as described in A. Antibodies used in ChIP assay were anti-TRβ, anti-NCoR, and anti-CBP. The specific region of the rGH TRE was q-PCR quantified (see Table 1 for primers). F, endogenous rGH mRNA expression in the presence of transfected TRβ or TRβ K443Q. T3 treatment is the same as described in A. G, WB shows the protein level of TRβ and TRβ K443Q in transfected cells from the experiment shown in E. *, indicate p < 0.05 compared with controls.
FIGURE 7.
Ligand-dependent release of corepressor from TRβ was disrupted by TRβ K443Q mutation. HepG2 cells were cotransfected with NCoR-FLAG and TRβ or TRβ K443Q. Cells were cotransfected with siRNAs and expression vectors as indicated in the figure. Coimmunoprecipitation was performed using affinity anti-FLAG resin in coimmunoprecipitation buffer (see “Experimental Procedures”). The immunoprecipitated complexes were detected in WB using anti-FLAG antibody (ab) to detect NCoR and anti-TRβ antibody. SUMO1 knockdown efficiency is shown in the WB in the lower panel.
FIGURE 8.
The influence of SUMO1 and SUMO3 on endogenous TSHβ mRNA expression and recruitment of endogenous TRβ and cofactors to the TSHβ nTRE. A, the endogenous T3-responsive TSHβ mRNA was determined using gene-specific primer (GSP2 and GSP3) for cDNA synthesis with cell culture and treatment conditions as described in the legend to Fig. 6. q-PCR primers GSP2 and GSP-reverse were used (see Table 1 for primer sequences). B, ChIP assays using GH3 cells. T3 treatment and assay conditions were the same as described in the legend to Fig. 6. *, indicates significance p < 0.05 compared with control.
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