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, 25 (6), 908-21

Differential Recruitment of Nuclear Coregulators Directs the Isoform-Dependent Action of Mutant Thyroid Hormone Receptors

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Differential Recruitment of Nuclear Coregulators Directs the Isoform-Dependent Action of Mutant Thyroid Hormone Receptors

Laura Fozzatti et al. Mol Endocrinol.

Abstract

Studies using mice deficient in thyroid hormone receptors (TR) indicate that the two TR isoforms, TRα1 and TRβ1, in addition to mediating overlapping biological activities of the thyroid hormone, T3, also mediate distinct functions. Mice harboring an identical dominant negative mutation (denoted PV) at the C terminus of TRα1 (Thra1(PV) mice) or β1 (Thrb(PV) mice) also exhibit distinct phenotypes. These knockin mutant mice provide an opportunity to understand the molecular basis of isoform-dependent functions in vivo. Here we tested the hypothesis that the distinct functions of TR mutant isoforms are directed by a subset of nuclear regulatory proteins. Tandem-affinity chromatography of HeLa nuclear extracts showed that distinct 33 nuclear proteins including nuclear receptor corepressor (NCoR1) and six other proteins preferentially associated with TRα1PV or TRβ1PV, respectively. These results indicate that recruitment of nuclear regulatory proteins by TR mutants is subtype dependent. The involvement of NCoR1 in mediating the distinct liver phenotype of Thra1(PV) and Thrb(PV) mice was further explored. NCoR1 preferentially interacted with TRα1PV rather than with TRβ1PV. NCoR1 was recruited more avidly to the thyroid hormone response element-bound TRα1PV than to TRβ1PV in the promoter of the CCAAT/enhancer-binding protein α gene to repress its expression in the liver of Thra1(PV) mice, but not in Thrb(PV) mice. This preferential recruitment of NCoR1 by mutant isoforms could contribute, at least in part, to the distinct liver lipid phenotype of these mutant mice. The present study highlights a novel mechanism by which TR isoforms direct their selective functions via preferential recruitment of a subset of nuclear coregulatory proteins.

Figures

Fig. 1.
Fig. 1.
Identification of mutant TR isoform-associated proteins from HeLa cells by tandem-affinity chromatography and proteomic analysis. A, Nuclear extracts prepared from control (lane 1), FH-αPV (lane 2), or FH-βPV (lane 3) were purified by tandem affinity chromatography as described in Materials and Methods. B, After peptide sequencing and proteomic analysis, the number of nuclear proteins associated with TR mutant isoforms is shown.
Fig. 2.
Fig. 2.
Physical interaction of NCoR1 with TR mutant isoforms in FH-βPV and FH-αPV cells. A, FH (controls) (lanes 1 and 4), FH-βPV (lanes 2 and 5), and FH-αPV (lanes 3 and 6) were cultured as described in Materials and Methods. Nuclear and cytoplasmic fractions were analyzed by Western blotting for the expression of markers for nuclear (PARP) (a) and cytoplasmic (α-tubulin) (b) fractions. (B-a) TRβ1PV and TRα1PV proteins were detected by monoclonal anti-Flag antibody, M2 (0.5 μg/ml), or by monoclonal anti-PV-specific antibody (2 μg/ml). B-b, Lanes 1 and 4 were from control cells as negative controls, indicating the specific bands detected in lanes 5 and 6. C, Association of NCoR1 and other nuclear proteins with TR mutants in FH-βPV and FH-αPV cells shown by coimmunoprecipitation. Nuclear extracts (1 mg) were immunoprecipitated with mouse anti-Flag M2 affinity gel followed by Western blot analysis with anti-NCoR1 antibody (C-a), anti-EIF4B antibody (C-b), anti-HSPA1B antibody (C-c), anti-TBL1XR1 antibody (C-d), anti-XRCC5 antibody (C-e), or anti-XRCC6 antibody (C-f). Lanes 5 and 6 were from nuclear extracts of FH-βPV and FH-αPV, respectively. Direct Western blot analysis (25 μg of nuclear extracts) is shown in lanes 1–3 as marked. IP, Immunoprecipitation.
Fig. 3.
Fig. 3.
Differential repression of mRNA expression of the C/ebpα gene in the liver of Thra1PV and ThrbPV mice. Quantitative real-time RT-PCR was performed as described in Materials and Methods to determine the mRNA expression of the C/ebpα gene in the liver of wild-type mice (bar 1), Thra1PV/+ mice (bar 2), ThrbPV/+ (bar 3), and ThrbPV/PV mice (bar 4). The data are expressed as mean ± ]scap]sem (n = 6; the P value is indicated). WT, wild type.
Fig. 4.
Fig. 4.
A, A schematic diagram of the proximal promoter of the mouse C/ebpα gene. This proximal promoter region is conserved between mouse and rat. It contains several cis-regulatory elements for several transcription factors as indicated. The binding sites of cis-regulatory elements are shown according to the relative position to the transcription start site. A TRE is located between nt −608 and −583. B, Binding of wild-type (lanes 1–4 for TRβ1; lanes 9–12 for TRα1) and mutant TR isoforms (lanes 5–8 for TRβ1PV and lanes 13–16 for TRα1PV) in the presence (lanes 4, 8, 12, and 16) or absence of NCoR1 3RIDs (lanes 3, 7, 11, and 15). Equal amounts of in vitro translated wild-type and mutant TR isoforms were used in the EMSA. C-a and C-b, More TRα1PV than TRβ1PV complexed with NCoR1RIDs on TRE. EMSA was carried out with increasing amounts of in vitro translated NCoR1RIDs with an equal amount of TRβ1PV (C-a) or TRα1PV (C-b). C-c, Comparison of the extent of remainder TR mutant binding as TRβ1PV/RXR and TRα1PV/RXR on mC/ebpα TRE. The band intensities in C-a and C-b were quantified by using ImageJ software and then graphed. NF-kB, Nuclear factor-κB.
Fig. 5.
Fig. 5.
The T3-dependent C/EBPα promoter activity was more inhibited by TRα1PV than by TRβ1PV in HepG2 cells stably expressing TRα1. HepG2TRα1 cells were cotransfected with 1 μg of the reporter plasmid (human C/EBPα promoter luciferase), 0.5 μg of RXRβ, and increasing concentrations of expression plasmid for TRα1PV (pcDNA3.1TRαPV) or TRβ1PV (pcDNA3.1TRβPV) as marked. Cells were treated without or with T3 (100 nm) for 24 h. Data were normalized against the protein concentration of the lysates. The percentage of luciferase activity after T3 treatment was calculated and defined as 100%. Assays were performed in triplicate and the data are expressed as mean ± ]scap]sem (n = 6). The data are expressed as bar graph (A) and x-y graph (B). The differences in the transcriptional activity were significant (*, P < 0.05; **, P < 0.01).
Fig. 6.
Fig. 6.
NCoR1 is more avidly recruited by TRα1PV than by TRβ1PV to the promoter of the C/ebpα gene. Primary mouse hepatocytes were prepared as described in Materials and Methods and infected with adenovirus encoding F-TRα1PV (lane 2, A-a) and F-TRβ1PV (lane 3, A-a), respectively. A-a, Identical amount of TRα1PV and TRβ1PV were expressed in hepatocytes. A-b, ChIP assay was carried out using anti-Flag antibody (A-b, lanes 3 and 6) or anti-NCoR1 antibody (PHQQ) (B-a, lanes 3 and 6) or IgG as controls (A-b and B-a, lanes 2 and 5) as described in Materials and Methods. Lanes 1 and 4 in A-b and B-a are input control. Shown is a representative result of three experiments. A-c, The intensities of bands shown in panels b of A and panel a of B were quantified by densitometry analysis, and data are shown as reference to the intensity of IgG as 1. The negative controls of using IGX1A (control primers) are also shown. B-c, The expression of C/ebpα mRNA was more repressed in hepatocytes expressing TRα1PV than expressing TRβ1PV (lanes are as marked). Mouse primary hepatocytes were tranduced with adenovirus encoding Flag-tagged TRα1PV, TRβ1PV, or control virus as shown in Fig. 6A-a. Total RNA (200 ng) prepared as described in Materials and Methods was used in real-time quantitative PCR analysis. Glyceraldedyde 3-phosphate dehydrogenase primer was used as an internal control. Data are expressed as mean ± sem (n = 4). NS, Nonsignificant (P = 0.3094).

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